Counterselection of GATC sequences in enterobacteriophages by the components of the methyl-directed mismatch repair system

Summary Weak to severe deficit of GATC sequences in the DNA of enterobacteriophages appears to be correlated with their undermethylation during growth indam ⁺ (GATC ade-methylase) bacteria. This observation is corroborated by the sequence analysis showing no evidence for site-specific mutagenicity of 6meAde. The MutH protein of the methyl-directed mismatch repair system recognizes and cleaves the undermethylated GATC sequences in the course of mismatch repair. To enquire whether the MutH function of the methyldirected mismatch repair system participates in counterselection of GATC sequences in enterobacteriophages, we have studied the yield of bacteriophage X174 containing either 0, 1, or 2 GATC sequences, in wild type,dam, andmut (H, L, S, U) Escherichia coli. Following transfection with unmethylated DNA containing two GATC sequences, a net decrease in the yield of infective particles was observed in all bacterialmutH ⁺ dam^– strains, whereas no detectable decrease was observed in bacteria infected by DNA without GATC sequence. This effect of the MutH function is maximum in wild type andmutL andmutS bacteria whereas the effect is not significant inmutU bacteria, suggesting an interaction of the, helicase II with the MutH protein.However, indam ⁺ bacteria, the presence of GATC sequences leads to an increased yield of infective particles. The effect of GATC sequence and its Dam methylation system on phage yield inmutH ^– bacteria reveals that methylated GATC sequences are advantageous to the phage. These results suggest that the methyl-directed mismatch repair system, and in particular its MutH protein, may have participated in severe counterselection of GATC sequences from enterobacteriophages, presumably, by DNA cleavage or by interfering with DNA replication or packaging when GATC sequences are undermethylated. Coevolution of the Dam and MutH proteins could then account for the loss of GATC sequences from DNA of bacteriophages growing indam ⁺ hosts.     

Regulatory mechanism of the tryptophan operon in Escherichia coli: possible interaction between trpR and trpS gene products

Summary The trpS5 mutation (a mutation in the structural gene for tryptophanyl-tRNA synthetase (TRSase) in E. coli), when present in the genetic background of strain KY913 (HfrH), results in the failure to grow at high temperature (42° C) in a complete medium. The rel (RC relaxed) marker present in this strain was found to be partly responsible for this temperature sensitivity. TRSase in such a strain was rapidly inactivated during growth at 42° C in rich media, but not in minimal media or in the presence of chloramphenicol. A partial derepression of anthranilate synthetase formation took place in the presence of excess tryptophan at growth-restricting temperatures. When some of the trpR mutations (including amber mutations) were combined with trpS5, the resulting double mutants (trpR trpS5) were temperature-insensitive, and TRSase was not inactivated at high temperature, in contrast to the trpR ⁺trpS5 strain. This effect of trpR mutations on temperature sensivity was shown not to be a secondary consequence of the constitutive expression of the trp operon. These findings suggest that the trpR ⁺ product interacts with the TRSase of the trpS5 mutant so as to bring about the growth-dependent inactivation of the enzyme. Furthermore, a special class of trpR mutants was obtained whose constitutivity with respect to the trp operon is manifested only in strains carrying trpS5 (but not trpS ⁺) grown at high temperatures. It is proposed that TRSase participates in repression trrough direct interaction with the product of the trpR gene.     

The use of specialised transducing phages in the amplification of enzyme production

Two types of trp phages have been used as model systems to investigate ways of optimising the expression of bacterial genes from transducing phage genomes.Excellent yields of trp enzymes were achieved by infecting a trpR^– host with Q ^– or Q ^– Q ^– S ^– derivatives of trpAM1, which expresses its trp genese exclusively from the trp promoter. The five trp geneproducts constituted more than 50% of the total soluble protein of infected cells under these conditions, and an even higher proportion of the protein synthesized after infection. In a trpR ⁺ host, phage DNA replication was easily able to override tryptophan-mediated repression by titration of the trp repressor protein. N ^– derivatives of trp phages carrying the trp promoter were equally productive, while having the advantage of being much simpler to construct and propagate.     

Studies on possible genetic effects of microwaves in procaryotic and eucaryotic cells

Summary The biological effects of microwaves in the hyperfrequency range, 9.4 GHz, 17 GHz, and 70–75 GHz were investigated in bacteria and yeast. At power densities below 60 mW/cm² and SAR values not exceeding 28 mW/g no significant effects on survival of repair competent and deficient strains were observed inEscherichia coli andSaccharomyces cerevisiae. In addition, microwaves of 17 GHz did not induce mutations inE. coli B/r WP2trp ^– uvr ^– above the spontaneous level, and the induction of nuclear reversions, cytoplasmic petite mutations and mitotic recombination as well as the efficiency of sporulation was not affected in yeast.     

Expression of the tryptophan operon in merodiploids of Escherichia coli. I. Gene dosage, gene position and marker effects

Summary Under conditions of derepression,Escherichia coli K12 strains diploid for thetrp operon specify more than twice as much enzyme as a haploid. The disproportionate increase probably occurs because episomally carriedtrp genes tend to specify more enzyme than do chromosomal genes.Operons harboring the nonsense mutationtrpA2 or the missense mutationtrpBYS-101 specify less protein than do wild-type operons. This effect varies with operon location in the case oftrpBYS-101.In a homozygoustrp merodiploid A46/F A46 reversion totrp ⁺ occurs three times as frequently in episomal DNA as in chromosomal DNA. Thus, if the chromosome: Ftrp episome ratio inE. coli is one, as demonstrated by Helinski and co-workers, the rate of gene expression and the rate of mutation can vary and depends upon the location of the DNA within the cell.Supported by Grant AM-12150 from the National Institutes of Health. Journal Paper No. 3973 of Purdue Agricultural Experiment Station.     

Cell inactivation,mutation and DNA strand-break induction by γ-rays at very low temperatures

Cell inactivation, mutation and DNA strand-break induction by -radiation have been investigated at very low temperatures (–78° C, –196° C, and –268° C). InEscherichia coli Y _mel ,lacI ⁺ lacI ^– andSalmonella typhimurium TA102,his ^–his ⁺ dose-modifying factors determined for low radiation doses are similar for both mutation induction and cell inactivation. The sensitivity of repair-deficient strainsE. coli polA ^– andE. coli recA ^– was also reduced at low temperature to a comparable extent. This suggests that the lesions which are responsible for cell inactivation and mutagenesis could be strongly mutually related and/or that different types of lesions which are responsible for cell inactivation and mutation induction in bacteria are reduced at low temperature to the same or similar extent. Likewise, a lower yield of DNA strand breaks in plasmids irradiated at low temperature was observed.     

Transfer ofF′ fromEscherichia coli K 12 toEscherichia coli B and to strains ofParacolobacter andKlebsiella

The transfer of theF episome fromEscherichia coli K 12 toE. coli B,Paracolobacter andKlebsiella was studied. The frequency of transfer of the episomal markers toE. coli B was very low. The large majority ofE. coli B cells which had received the episomal markerslac ⁺ orgal ⁺ were F^–, which indicates that the episomal markers were stably integrated on the chromosome. Recombinants from K 12 F⁺ × B F^– crosses were mostly F^–. These results suggest that the multiplication of theF-factor ofE. coli K 12 is restricted inE. coli B. The transfer of theF-lac ⁺ Ad ⁺ episome fromE. coli K 12 toParacolobacter andKlebsiella strains was in most cases only possible when donor and acceptor strain were plated together on selective media. Stable incorporation of episomal markers was also found withParacolobacter coliforme. Paracolobacter aerogenoides andKlebsiella aerogenes strains could be infected withF-lac ⁺ Ad ⁺. The episomal markers were not incorporated and the episomes were easily lost, which indicates that these strains contained theF factor in the autonomous state.     

Effect of amino sugars on transformation of strainsStreptococcus Wicky andBacillus subtilis 168trp 2 −

The inhibitory effect ofd-glucosamine andd-galactosamine on the induction of competence inStreptococcus Wicky was detected. These sugars also inhibited the transformation inBacillus subtilis 168trp ₂ ^? . The same effect was observed inBacillus subtilis when usingN-acetyl-d-galactosamine.     

The influence of amino acid starvation on the UV reversibility of the strains ofEscherichia coli B/rthy − trp − Hcr + andEscherichia coli B/rthy − trp − Hcr −

The fraction of inducedtrp ⁺ reversions in the strains ofEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁺ andEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁻ was studied in the course of starvation for an essential amino acid. UV light as a mutagenic factor was used. It was found that there is a decrease in the proportion of inducedtrp ⁺ reversions in the strain ofHcr ⁺ type during starvation. Such a decrease was however observed only with that fraction oftrp ⁺ reversions which is expressed in selective plates where several divisions of irradiated cells are caused. The proportion oftrp ⁺ reversions expressed on minimal plates does not change during starvation. With the strain ofHcr ⁻ type the proportion of inducedtrp ⁺ mutations remains unaltered irrespective of the nature of the selective plates.     

Temperature optimization ofin vivo expression from theE. coli trp andtrp::lac promoters

Summary We have studied the effect of temperatures between 20°C and 40°C on the activity ofE. coli promoters. TheE. coli lac UV5,trp::lac, andtrp promoters reach their maximal activity at 37°C while thetet::trp promoter reaches its maximal activity at 40°C. The addition of 5g IAA/ml was sufficient to derepress thetrp promoter over the temperature range studied.     

Cloning the trpR gene.

Summary In Escherichia coli, the structural gene for purine nucleoside phosphorylase, deoD, is subject to insertional inactivation by prophage . From one such secondary site lysogen, strain SP265, one may isolate deletions that remove all or part of the trpR gene and other genes in the deo-thr sector of the E. coli chromosome. Specialized transducing phages harboring serB ⁺ and trpR ⁺ were liberated following induction of SP265. All such phages were N-defective, bio-type pseudolysogens whose DNA persisted in the form of plasmids. A collection of transducing phages, differing in their complement of bacterial DNA, was used to locate cleavage sites for bamHI, SalI, and PvuI within the deoD-trpR region of the E. coli genome. The trpR gene lies within a specific 950 base pair BamHI-PvuI segment.A 1250 base pair BamHI fragment carrying a functional trpR gene was cloned into the amplifiable plasmid pBR322. A single SalI site in this fragment was shown to lie within the trpR gene.In two situations where increased gene dosage might generate elevated amounts of Trp repressor (N-defective trpR ⁺ pseudolysogens and strains harboring pBR322 trpR ⁺ plasmids) neither tryptophan auxotrophy, enhanced sensitivity to DL-5-methyltryptophan, nor super repression of the tryptophan biosynthetic enzymes was observed.Journal Paper No. 7426 of the Purdue University Agricultural Experiment Station     

Methyl directed DNA mismatch repair inVibrio cholerae

Mismatches in DNA occur either due to replication error or during recombination between homologous but non-identical DNA sequences or due to chemical modification of bases. The mismatch in DNA, if not repaired, result in high spontaneous mutation frequency. The repair has to be in the newly synthesized strand of the DNA molecule, otherwise the error will be fixed permanently. Three distinct mechanisms have been proposed for the repair of mismatches in DNA in prokaryotic cells and gene functions involved in these repair processes have been identified. The methyl-directed DNA mismatch repair has been examined inVibrio cholerae, a highly pathogenic gram negative bacterium and the causative agent of the diarrhoeal disease cholera. The DNA adenine methyltransferase encoding gene (dam) of this organism which is involved in strand discrimination during the repair process has been cloned and the complete nucleotide sequence has been determined.Vibrio cholerae dam gene codes for a 21.5 kDa protein and can substitute for theEscherichia coli enzyme. Overproduction ofVibrio cholerae Dam protein is neither hypermutable nor lethal both in Escherichia coli andVibrio cholerae. WhileEscherichia coli dam mutants are sensitive to 2-aminopurine,Vibrio cholerae 2-aminopurine sensitive mutants have been isolated with intact GATC methylation activity. The mutator genesmutS andmutL involved in the recognition of mismatch have been cloned, nucleotide sequence determined and their products characterized. Mutants ofmutS andmutL ofVibrio cholerae have been isolated and show high rate of spontaneous mutation frequency. ThemutU gene ofVibrio cholerae, the product of which is a DNA helicase II, codes for a 70 kDa protein. The deduced amino acid sequence of themutU gene hs all the consensus helicase motifs. The DNA cytosine methyltransferase encoding gene (dam) ofVibrio cholerae has also been cloned. Thedcm gene codes for a 53 kDa protein. This gene product might be involved in very short patch (VSP) repair of DNA mismatches. The vsr gene which is directly involved in VSP repair process codes for a 23 kDa protein. Using these information, the status of DNA mismatch repair inVibrio cholerae will be discussed.     

Base pair substitutions caused by the uvr502 mutation affecting mutation rates and UV-sensitivity of Escherichia coli

Summary The types of base pair substitutions induced by the uvr502 mutator activity were studied using the isogenic uvr ⁺ and uvr502 strains bearing an ochre or missense mutations in the trp operon. It was found that the uvr502 mutation increased the frequency of both structural gene (true) reversions and suppressor mutations in the trp _oc ^– mutant. The trpA58 missense mutation was also reverted by the uvr502 allele and 5-methyl tryptophane resistant as well as 5-methyl tryptophane sensitive Trp⁺ revertants were formed. However the uvr502 mutation was unable to increase significantly the frequency of Trp⁺ revertants in the rpA78 mutant. With the help of key of Yanofsky et al. (1966b) and codon catalogue it could be concluded that the uvr502 mutation induces transitions in both directions but not A:TC:G and probably not G:CT:A transversions. Incubation of the uvr502 mutant with either of four deoxyribonucleosides has no effect on its spontaneous mutability while deoxyguanosine and deoxyadenosine reduce the mutagenic effect of 2-aminopurine in the uvr ⁺ strain, suggesting that the mutator effect of the uvr502 mutation has nothing to do with the formation of mutagenic base analogue or insufficient synthesis of bases.     

The segregation of the SbcA and Rac phenotypes in an Escherichia coli recB mutant

Summary In certain HfrxF^– recB ^– crosses recombinant progeny were examined for their SbcA and Rac phenotypes. Recombinants which inherited either his ⁺ or trp ⁺ from the donor in an Hfr recB21 sbcA8xF^– recB21 Rac^–SbcA⁺ cross acquired the RecB⁺ phenotype in most instances (presumably by inheriting the sbcA8 allele). Several independent Rec⁺ (sbcA8) recombinants from this cross were converted back to the Rec^– (sbcA ⁺) phenotype by mating with a Rac⁺ SbcA⁺ Hfr. Ten out of 14 of these Rec^– recombinants retained the Rac^– phenotype of the original parent. It was concluded that these results were inconsistent with the hypothesis that sbcA is a gene carried by a Rac prophage.     

Studies on two classes of rifampicin-resistant mutants of the nitrogen-fixing cyanobacteriumNostoc muscorum

Two types of nitrosoguanidine-induced rifampicin-resistant mutants ofNostoc muscorum were isolated and characterized. Compared with the wild type, the strainrif-1 (rif ^r het ⁺ nif ⁺ blu) revealed high growth rate, heterocyst frequency (10%–12%), nitrogenase activity, phycocyanin pigment, photosynthetic O₂ evolution, and higher activities of phosphoribulokinase and Fd-NADP⁺-oxidoreductase. The heterocyst spacing pattern in the mutant was altered and did not respond to 7-azatryptophan, -2-thienylalanine, rifampicin, andl-methionine-dl-sulfoximine treatment. The second type of mutantrif ^r -2 (rif ^r het ⁺ nif ^–) did not show nitrogenase activity and was unable to grow on molecular nitrogen even under microaerobic conditions, although it produced heterocysts (6%–7%) under these conditions of incubation. The pattern of macromolecular synthesis, particularly of RNA and protein, the rate of acetylene reduction, and photosynthetic O₂ evolution were not affected in the mutant strains with the treatment of drug. The characteristics of the mutants reflected the possibility of pleiotropic mutation and also suggested that therif marker is most likely associated in close genetic proximity with regulatory gene(s) ofhet andnif system.     

Kinetic mechanism of Na+, K+, Cl−-cotransport as studied by Rb+ influx into HeLa cells: Effects of extracellular monovalent ions

Summary Ouabain-insensitive, furosemide-sensitive Rb⁺ influx (J _Rb) into HeLa cells was examined as functions of the extracellular Rb⁺, Na⁺ and Cl^– concentrations. Rate equations and kinetic parameters, including the apparent maximumJ _Rb, the apparent values ofK _m for the three ions and the apparentK _i for K⁺, were derived. Results suggested that one unit molecule of this transport system has one Na⁺, one K⁺ and two Cl^– sites with different affinities, one of the Cl^– sites related with binding of Na⁺, and the other with binding of K⁺(Rb⁺). A 11 stoichiometry was demonstrated between ouabain-insensitive, furosemidesensitive influxes of²²Na⁺ and Rb⁺, and a 12 stoichiometry between those of Rb⁺ and³⁶Cl^–. The influx of either one of these ions was inhibited in the absence of any one of the other two ions. Monovalent anions such as nitrate, acetate, thiocyanate and lactate as substitutes for Cl^– inhibited ouabain-insensitive Rb⁺ influx, whereas sulfamate and probably also gluconate did not inhibitJ _Rb. From the present results, a general model and a specialized cotransport model were proposed: 1) In HeLa cells, one Na⁺ and one Cl^– bind concurrently to their sites and then one K⁺ (Rb⁺) and another Cl^– bind concurrently. 2) After completion of ion bindings Na⁺, K⁺(Rb^–) and Cl^– in a ratio of 112 show synchronous transmembrane movements.     

Voltage dependence of current through the Na,K-exchange pump ofRana oocytes

Summary We have studied current (I _Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I _Str was measured as the strophanthidin-sensitive current in the presence of Ba^2–, Cd²⁺ and gluconate (in place of external Cl^–). In addition,I _Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK _0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na⁺-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI _Str depended directly onV _m up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na⁺] markedly decreased the voltage sensitivity ofI _p , while producing only a small change in the maximal outwardI _Str. In contrast, decreasing the external [K⁺] from 25 to 2.5mm reducedI _Str at 0 mV without substantially affecting its voltage dependence. At K⁺ concentrations of 1mm, both the absolute value ofI _Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI _Str by [K⁺] _o over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K⁺ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K⁺] _o >1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na⁺ and K⁺ are consistent with results obtained in other tissues and may reflect an ion-well effect.     

Characterization of sodium currents in mammalian sensory neurons cultured in serum-free defined medium with and without nerve growth factor

Summary The influence of nerve growth factor (NGF) on Na currents of rat dorsal root ganglia (DRG) was studied in neurons obtained from newborns and cultured for 2–30 hr inserum-free defined medium (SFM). Cell survival for the period studied was 78–87% both with and without NGF. Na currents were detected in all cells cultured for 6–9 hr. They were also detected after 2 hr in culture in 21.5% of the cells cultured without NGF (–NGF cells), and in 91.5% of the cells cultured with NGF (+NGF cells). Current density of the -NGF cells was 2.3 and 2 pA/m² after growth for 2 and 6–9 hr, respectively, compared to 3.0 and 3.9 pA/m² for the +NGF cells. The +NGF cells were separated into fast (F), Intermediate (I) and slow (S) cells, based on the Na current they expressed, while -NGF cells were all of theI type.F, I andS currents differed in their voltage-dependent inactivation (Vh ₅₀=–79, –28 and –20 mV), kinetics of inactivation (tau _h =0.55, 1.3 and 7.75 msec), and TTX sensitivity (K _i=60, 550 and 1100nm). All currents were depressed by [Ca] _o with aKd _Ca of 22, 17 and 8mm forF, I andS currents, respectively. Current density ofF andS currents was 5.5 and 5 pA/m² for theI current. The concentration-dependent curve ofI currentvs. TTX indicated thatI current has two sites: one withF-like and another withS-likeK _i for TTX. Hybridization ofF andS currents yieldI-like currents. Thus, the major effect of NGF on Na currents in SFM is the accleration of Na current acquisition and diversity, reflected in an increase of either theS orF type in a cell.     

Expression of Escherichia coli tryptophan operon in Rhizobium leguminosarum.

Summary RP4-trp hybrid plasmid containing Escherichia coli whole tryptophan operon was conjugatively transferred from E. coli to Rhizobium leguminosarum strains carrying mutations in different trp genes, converting their Trp^– phenotype to Trp⁺. That the phenotype change of the R. leguminosarum cells was due to the presence of the E. coli tryptophan operon was verified by the isolation of RP4-trp hybrid plasmid from the R. leguminosarum conjugant cells, and by re-transfer of RP4-trp plasmid by conjugation back to E. coli trp and Pseudomonas putida trp strains. Enzymatic activities of anthranilate synthetase and subunit of tryptophan synthetase in crude extracts of R. leguminosarum cells containing RP4-trp plasmid were much higher than that of the wild-type cells and were not repressed by the presence of tryptophan in the culture medium.     

Establishment of a novel sensitive method for detecting methylation modification on DNA of escherichia coli cell

This study focused on finding a novel sensitive method to determine the methylation modification at DNA dam (GATC) sites in Escherichia coli. A new plasmid which contained three GATC sites recognized by restriction enzyme BclI and one GAATTC site recognized by EcoRI was transformed into E. coli stains AB1157(dam ⁺) and GM2929(dam ⁻) respectively. Then the plasmid DNA was digested by restriction enzyme BclI(T*GATCA), which was sensitive to methylation. The results showed that the plasmid derived from AB1157 was not digested while that from GM2929 was, for the methylation level of the former was high while the latter was low. So by detecting the methylation of plasmid transferred into the strain, we could determine whether methylaion existed at DNA dam (GATC) site in E. coli. This method was effective and rapid; moreover, the digested fragments were not dispersive. It also made a basis for the detection of whether methylation occurred in mode beings by low-energy ion beam. The article is published in the original.